This project will investigate the mechanisms of NaCl absorption by airway surface epithelium in order to determine if and how the Na and Cl levels of normal ASL may be maintained at values markedly less than plasma. Our preliminary data show that forskolin stimulates, and NPPB inhibits, absorption of liquid across primary cultures of bovine and human tracheal epithelium, without affect transepithelial mannitol fluxes. These results strongly suggest that a substantial fraction of absorbed Cl passes by a transcellular route. In CF, the absence of functional CFTR in the apical membrane should block this route for Cl absorption and promote higher than normal levels of Na and Cl in ASL. This project will determine whether the appropriate driving forces and permeation pathways for movement of Cl exist to enable non-CF surface epithelium to maintain low NaCl concentrations in ASL. First, we will measure the electrochemical driving force for Cl movement across the apical membrane over a wide range of mucosal NaCl concentrations. With 120 mEq/l of mucosal Cl, preliminary microelectrode data on bovine tracheal epithelium show clearly that there is a large driving force for Cl entry across the apical membrane. The mucosal NaCl concentration at which this driving force reverse should be close to the minimal concentration of NaCl that can be maintained in ASL. Second, by measuring changes in nasal PD in response to activators and blockers of CFTR, we will obtain indirect information on the direction of the electrochemical driving force for Cl movement across apical membrane of human airways in vivo. Third, the regulation, pharmacology and biophysics of a newly discovered basolateral cAMP-activated Cl channel will be determined, and we will elucidate how opening of apical and basolateral Cl channels is coordinated.